Inhibitory effects of cinnamaldehyde on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung carcinogenesis in rasH2 mice.

Previously we reported a lack of modification by cinnamaldehyde (CNMA) of development of lung proliferative lesions induced by urethane in CB6F1-TgHras2 (rasH2) mice. In the present study, we re-evaluated CNMA effects using the same rasH2 strain and non-transgenic littermates initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Sixteen mice/strain/sex received intraperitoneal NNK injections at a dose of 3 mg/mouse once a week for 2 weeks followed by free feeding of commercial diet containing 5000 ppm CNMA for 26 weeks. Additional groups were maintained without NNK injection and/or CNMA feeding for 28 weeks. Lung tumors were induced by NNK in both rasH2 and non-Tg males and females at incidence ranging from 63 to 100%. CNMA treatment significantly reduced the combined incidence of adenomas and carcinomas from 86 to 31% in rasH2 males (P<0.05), but no significant influence was evident in females. The multiplicity of NNK-induced lung tumors was also significantly reduced in rasH2 males given CNMA (P<0.01). Similar effects were also observed in non-Tg females given CNMA after NNK initiation. The results of our study strongly indicate that CNMA is capable of inhibiting development of NNK-initiated pulmonary tumorigenesis in rasH2 and non-Tg mice.     

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in heterozygous p53-deficient female mice at the same dose in a 6-month study. To examine whether phenolphthalein carcinogenic potential can be detected in male and female transgenic (Tg) mice carrying the human c-Ha-ras gene (rasH2 mice) and their wild-type littermates (non-Tg mice), a diet containing 3000, 6000 or 12000 ppm was given for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that rasH2 mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.     

Carcinogen dose-dependent variation in the transgene mutation spectrum in urethane-induced lung tumors in transgenic mice carrying the human prototype c-Ha-ras gene

Urethane-induced lung tumors and their genetic changes were investigated in transgenic (Tg) mice carrying a human prototype c-Ha-ras gene (rasH2 mice). Male and female rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1000 mg/kg of urethane once or three times at 2-day intervals. Hyperplasias and adenomas of the lung were observed in all animals of each group from week 10, and carcinomas were observed in male and female rasH2 mice of the triple injection group from week 10 and female non-Tg mice of the single injection group at 15/20 weeks. The multiplicities of lung proliferative lesions including hyperplasias, adenomas and carcinomas, in treated rasH2 mice were significantly higher than those in treated non-Tg mice. CAG to CTG transversions were observed in the c-Ha-ras gene in these lung proliferative lesions of rasH2 mice of the single injection group at high incidence (male: 58.3%, female: 62.5%), but no mutations of the mouse c-Ki-ras gene were evident in either rasH2 or non-Tg mice. In the triple injection group, transgene mutations were detected at a relatively low incidence, and mouse c-Ki-ras gene mutations(CAA to CGA) were observed in both rasH2 and non-Tg mice. These results suggest that the variation of the lesions induced by different doses of urethane was not the cause of the variation of the mutation spectrum and mutations of both transgene and mouse c-K-ras gene are not principal genetic events in urethane-induced lung proliferative lesions in rasH2 mice.     

A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies

We investigated the susceptibility of 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in male CB6F1-Tg-rasH2 @Jcl mice (Tg mice). The Tg mice were administered 4-NQO (20 p.p.m. in drinking water) for 2, 4, 6 or 8 weeks, and thereafter they were untreated up to week 24. At week 24, a higher incidence (80%) of tongue neoplasm with dysplasia was noted in the mice that received 4-NQO for 8 weeks in comparison with the other groups (20% incidence for each) treated with 4-NQO for 2, 4 and 6 weeks. Esophageal tumors also developed in the Tg mice were 4-NQO. Immunohistochemical observation revealed that the EP receptors, especially EP(1) and EP(2), expressed in the tongue and esophageal lesions induced by 4-NQO, thus suggesting the involvement of prostaglandin (PG) E(2) and EP(1,2) receptors in the tongue and esophageal carcinogenesis. Using this animal model, we investigated the potential chemopreventive ability of pitavastatin (1, 5 and 10 p.p.m. in diet for 15 weeks), starting 1 week after the cessation of 4-NQO-exposure (20 p.p.m. in drinking water for 8 weeks). Dietary pitavastatin at 10 p.p.m. significantly reduced the incidence and multiplicity of the tongue, but not esophageal neoplasms by the modulation of prostaglandin E2 biosynthesis, EP(1) and EP(2) expression and proliferation. Our results thus suggest that a rasH2 mouse model of 4-NQO-induced tongue and esophageal carcinogenesis can be utilized for investigating the pathogenesis of cancer development in these tissues and may well prove to be useful for identifying candidate cancer chemopreventive agents for the upper digestive organs.     

Decreased c-kit function inhibits enhanced skin carcinogenesis in c-Ha-ras protooncogene transgenic mice

We previously showed that rasH2 transgenic mice carrying the human c-Ha-ras protooncogene are highly susceptible to chemical skin carcinogenesis. In the dermis of rasH2 mice, mast cells are recruited constitutively, and the number of mast cells increases more than in wild-type mice in response to treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. To determine whether enhanced skin tumor development in rasH2 mice is dependent on the recruitment of mast cells, we generated rasH2 KIT(W/Wv) mice by crossing rasH2 mice and W or W(v) KIT mutants, and examined the chemical skin carcinogenesis. In rasH2 KIT(W/Wv) mice, mast cells were not found in the dermis either before or after treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Papilloma multiplicity was up to 4.6-fold higher in rasH2 KIT(+/+) mice compared with their rasH2 KIT(W/Wv) siblings. At 12 weeks after the experiment began, the volumes of tumors were significantly smaller in rasH2 KIT(W/Wv) relative to rasH2 KIT(+/+) mice (rasH2 KIT(W/Wv): 29.2 +/- 19.9 mm(3) versus rasH2 KIT(+/+): 179.6 +/- 726.6 mm(3); P = 0.0153). There was no difference in the latency or multiplicity of papillomas between mice without the rasH2 transgene, KIT(W/Wv) mice and their wild-type littermates. Western blot analysis showed that expression of H-RAS protein in the skin was equivalent in rasH2 KIT(W/Wv) and rasH2 KIT(+/+) mice. In conclusion, the inhibition of c-kit decreased H-ras-induced skin carcinogenesis. The suppression of c-kit may be a unique and effective target as a preclinical model of cancer treatment where the activation of H-ras has a significant role. Targeting mast cells could also be a potential strategy for treating malignancies.     

Hepatocarcinogenicity of chlordane in B6C3F1 and B6D2F1 male mice: evidence for regression in B6C3F1 mice and carcinogenesis independent of ras proto-oncogene activation

Logistic regression analysis of age-specific prevalences forneoplastic and non-neoplastic liver lesions was used to examinetreatment responses for B6C3F1 and B6D2F1 male mice continuouslyexposed to chlordane (55 p.p.m.) and to determine whether neoplasmswere dependent on continuous exposure in the B6C3F1 mice. Inorder to determine if ras oncogene activation plays a role inthe carcinogenicity of chlordane and whether the activationis dependent on genetic background, liver tumors from chlordane-treatedB6C3F1 and B6D2F1 mice were analyzed for the presence of activatingmutations in the ras oncogene. The overall liver tumor prevalenceat terminal killing was nearly 100% for both strains; however,the age-specific prevalence increased more rapidly in B6C3F1mice than in B6D2F1 mice. Tumor-bearing B6C3F1 mice had an averageof two more tumors per liver than B6D2F1 mice at their respectiveterminal killings (5.4 versus 3.3). When chlordane exposurewas discontinued for a group of B6C3F1 mice (‘stop’group) at 491 days of age, overall tumor multiplicity significantlydecreased by 30% from an average of 4.4 per tumor-bearing-animalat 525 days to 3.1 at terminal killing (568 days). Over thesame time period the prevalence of hepatocellular carcinomassignificantly decreased from 80 to 54% and adenomas from 100to 93% by terminal killing in B6C3F1 ‘stop-group’mice. Chlordane induced diffuse hepatocellular centrilobularhypertrophy, frequent multinucleate hepatocytes, toxic changeand hepatoproliferative lesions composed predominantly of acidophilichepatocytes in nearly 100% of both the B6C3F1 and B6D2F1 mice.The development of histological evidence of toxicity closelyparalleled the temporal development of hepatocellular neoplasiaand decreased in severity when the tumor burden was maximal.No H- or K-ras mutations were detected in the chlordane-inducedhepatocellular tumors in B6C3F1 mice (15 adenomas and 15 carcinomas)or B6D2F1 mice (10 adenomas and 10 carcinomas). In conclusion,chlordane induced liver tumors in both B6C3F1 and B6D2F1 malemice by mechanisms independent of ras oncogene activation and30% of both benign and malignant liver tumors in the B6C3F1mice regressed after exposure was discontinued.     

Hepatic tumors induced by carbon tetrachloride in transgenic mice carrying a human c-H-ras proto-oncogene without mutations

Hepatic tumors were generated in mice by repeated administration of carbon tetrachloride (CCI₄). Eight transgenic (Tg) mice carrying a human c-H-ras proto-oncogene (rasH2 line) and 9 non-Tg mice were killed at 20 weeks. Tg mice developed more tumors than did non-Tg littermates. Most tumors were neoplastic nodules, but I hepatocellular carcinoma (HCC) was found in a Tg mouse at 20 weeks. Three Tg and 2 non-Tg mice were kept without further administration of CCI₄. Two Tg mice died at 30 weeks of HCC with intra-abdominal bleeding, and I Tg mouse developed HCC with a mesenteric metastasis at 32 weeks. No HCC was found in 2 non-Tg mice at 32 weeks. Although mutations at codon 12, 13, and 61 of the H-ras gene are often found in murine hepatocarcinogenesis, neither the tumors, including one HCC, nor the normal cells revealed any such mutations. These results showed that the unmutated human c-H-ras gene facilitates malignant transformation of hepatocytes when continuous liver-cell death and regeneration is caused by repeated administration of CCI₄. © 1994 Wiley-Liss, Inc.     

The possible mechanism of enhanced carcinogenesis induced by genotoxic carcinogens in rasH2 mice

Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.     

Rapid induction of uterine endometrial proliferative lesions in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) given a single intraperitoneal injection of N-ethyl-N-nitrosourea

In our previous study, uterine endometrial stromal sarcomas and atypical hyperplasias of the endometrial glands were induced in heterozygous p53 deficient mice (p53 (+/-) mice) of the CBA strain given a single dose of N-ethyl-N-nitrosourea (ENU). In order to clarify whether uterine tumors can be induced in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) that are very susceptible to genotoxic carcinogens, rasH2 mice and their wild-type littermates received an intraperitoneal injection of 120 or 0mg/kg body weight of ENU followed by no further treatment for 22 weeks. Eighteen and 94% of ENU-treated rasH2 mice had uterine endometrial adenocarcinomas and atypical hyperplasias, respectively. Other malignant and benign tumors such as lung alveolar/bronchiolar adenomas and carcinomas, forestomach squamous cell papillomas and carcinomas, splenic hemangiomas/sarcomas, skin papillomas, malignant lymphomas and harderian gland adenomas were also observed in ENU-treated rasH2 mice. The result in the present study suggests that female rasH2 mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine epithelial tumors.     

Molecular analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice following inhalation exposure to 1,3-butadiene

Chronic exposure to 1,3-butadiene (BD) results in early occurrenceand high incidence of lethal lymphomas in male B6C3F1 mice.Male B6C3F1 lacI transgenic mice (BigBlue^TM) were exposed byinhalation to 0, 62.5, 625 or 1250 p.p.m. BD for 4 weeks (6h/day, 5 days/week). The lacI^- mutant frequency and mutationalspectrum were evaluated in DNA isolated from bone marrow cells.Two weeks after exposure the lacI^- mutant frequency in bonemarrow from BD-exposed mice had increased 2- to 3.5-fold overair control mice. DNA sequence analysis of 56 and 54 lacI^- mutantsfrom the air control and butadiene-exposed groups, respectively,demonstrated that there was a shift in the spectrum of basesubstitution mutations at A:T sites in BD-exposed mice (6/26)compared to the air control mice (2/45). A:T     

Increased frequency of K-ras mutations in lung neoplasms from female B6C3F1 mice exposed to ozone for 24 or 30 months

The National Toxicology Program recently completed long-termozone inhalation studies in B6C3F1 mice and F344/N rats. Miceand rats were exposed to 0, 0.5 or 1.0 p.p.m. ozone by inhalationfor 24 or 30 months. There was an increased incidence of lungneoplasms in B6C3F1 mice. However, there was no evidence ofcarcinogenicity in F344/N rats. The objectives of this studywere to (i) evaluate benign and malignant lung neoplasms fromB6C3F1 mice for mutations in the K-ras gene at codons 12, 13and 61, (ii) determine if the frequency and spectra of K-rasmutations were unique for ozone-induced lung neoplasms, (iii)determine if specific K-ras mutations were associated with thesize and morphological patterns of lung neoplasms or ozone exposureconcentrations and (iv) screen lung neoplasms by immunohistochemicalmethods for the p53 protein. K-ras mutations were detected bysingle-strand conformation analysis and identified by directsequencing of polymerase chain reaction-amplified DNA isolatedfrom formalin-fixed, paraffin-embedded neoplasms. K-ras mutationswere detected in 73% of ozone-induced neoplasms, as comparedwith 33% of lung neoplasms from controls. The predominant mutationsconsisted of A     

Further genetic analyses of skin tumor promoter susceptibility using inbred and recombinant inbred mice

To explore further the genetics of susceptibility of skin tumorpromotion in inbred mice, several aspects of responsivenessto 12-O-etradecanoylphorbol-13-acetate (TPA) were examined inC3H/He mice and segregating crosses between this mouse strainand C57BL/6 mice as well as BXD and BXH recombinant inbred (RI)strains. Dose-response relationships were established for skintumor promotion by TPA following initiation with 7, 12-dimethylbenz(a)anthracenein C3H/He and B6C3F1, as well as several other mouse stocksand strains included for comparison. The relative responsivenessto TPA skin tumor promotion was: SENCAR > > DBA/2 >C3H/He = B6D2F1 > B6C3F1 > > C57BL/6. Analyses of thesusceptibility of B6C3F2 and B6C3F1 x C57BL/6 backcross micesuggested that a minimum of two dominant genetic loci controlresponsiveness to phorbol ester promotion in these mice. Furtheranalysis of BXH and BXD RI strains suggested the presence offour distinct promotion-responsive phenotypes controlled bya minimum of two genetic loci. The existence of a ‘hyper-responsive’phenotype in the sets of RI strains, however, suggests thata third, recessive locus also may play a role in controllingresponsiveness to TPA, promotion. At 48 h after the last offour applications of TPA, marked hyperplasia and an increasein dark basal keratino-cytes were observed in C3H/He mice, whereasin B6C3F1 mice the response in these parameters was intermediatebetween C3H/He and C57BL/6 mice. A marked dermal inflamation,as determined by infiltration of polymorpho-nuclear cells, wasobserved in C3H/He and B6C3F1 mice, whereas little was notedin C57BL/6 mice. Furthermore, histological evaluations of selectedBXD RI strains revealed a significant correlation between themagnitude of the hyperplasia response and the percentage ofmice bearing tumors. The present data, in conjunction with ourprevious studies, confirm that the major gene(s) controllingsusceptibility to tumor promoter induced by TPA in two sensitivestrains (i. e. DBA/2 C3H/He) are similar of closely linked tothose for induction of sustained hyperplasia. In addition, thepresent data provide new evidence for a model where allelicdifferences at a minimum of three loci contribute to geneticdifferences in susceptibility to phorbol ester promotion inDBA/2 and C3H/He versus C57BL/6 mice.     

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two majorby-products formed during chlorine disinfection of drinkingwater, increase the incidence of tumors in B6C3F1 mice by 6-and 3-fold respectively. In order to understand better the mechanismby which these two compounds induce liver tumors, the incidenceand spectrum of mutations in the K- and H-ras proto-oncogenesin these tumors were analyzed. DNA from spontaneous, DCA- andTCA-induced liver tumors from B6C3F1 male mice was evaluatedfor point mutations in exons 1, 2 and 3 of the two genes bysingle-stranded conformation polymorphism. Results demonstrateda similar incidence of mutations for exon 2 of H-ras in spontaneouscarcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%),1.0 g/1(48%) and TCA 4.5 g/l (45%). Only four samples showedmutations in the other exons of H-ras or in K-ras. Sequenceanalysis of spontaneous tumor samples with second exon H-rasmutations revealed a change in codon 61 from CAA to AAA in 80%and CAA to CGA in 20% of tumors. In contrast, tumors with H-rasmutations from DCA-treated mice revealed a H-61 change fromCAA to AAA in 21 % at 3.5 g/l and 16% at 1.0 g/l. CAA to CGAwas observed in 50% of tumors from mice given DCA 3.5 or 1.0g/l, and CAA to CTA was present in 29% and 34% of the two dosagegroups respectively. Interestingly, TCA showed the same mutationalspectrum as the spontaneous liver tumors. The data indicatesthat induction of liver carcinoma by DCA and TCA involves activationof the H-ras proto-oncogene at a frequency similar to that observedin spontaneous tumors. However, the mechanism(s) for inducinghepatocellular carcinoma does not appear to be identical forDCA and TCA.     

Occurrence of tumor antigen pRL1a specific CD8 T cells in spleen cells from syngeneic BALB/c, semiallogeneic (BALB/c x C57BL/6)F1 and allogeneic C57BL/6 mice

We investigated the generation of CD8 cytotoxic T-lymphocytes (CTL) that recognized a dominant pRL1a peptide bound to H-2L(d) molecule on RL male 1 leukemia in spleen cells from RL male 1-bearing syngeneic BALB/c, semiallogeneic CB6F1 and allogeneic B6 mice by repetitive in vitro stimulation with RL male 1 tumor. CD8 T cells in cultures were also analyzed by H-2L(d)/pRL1a tetramer staining. We showed that pRL1a-specific CTL were more efficiently generated in spleen cells from RL male 1-bearing high responder CB6F1 mice than in low responder BALB/c mice, and this correlated well with the occurrence of H-2L(d)/pRL1a tetramer binding CD8 T cells. Furthermore, we showed that in spleen cells from RL male 1-bearing allogeneic B6 mice, H-2L(d)/pRL1a complex specific CD8 T cells were present at a significant frequency. H-2L(d)/pRL1a recognizing B6 CTL but not BALB/c or CB6F1 CTL gradually lost CD8 expression on their surface by multiplication of in vitro stimulation.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

The Hcr (hepatocarcinogen resistance) loci of DBA/2J mice partially suppress phenotypic expression of the Hcs (hepatocarcinogen sensitivity) loci of C3H/HeJ mice

Both male DBA/2J and C3H/HeJ mice are highly susceptible tohepatocarcinogenesis induced by experimental treatment withN,N-diethylnitrosamine (DEN) relative to male C57BL/6J mice.While C3H/HeJ mice carry multiple sensitivity loci, designatedHcs (hepatocarcinogen sensitivity), our previous study indicatedthat the susceptibility of DBA/2J mice results from the combinedeffects of multiple sensitivity loci and two major resistanceloci, Hcr-1 and -2 (hepatocarcinogen resistance). We proposedthat BXD-15 recombinant inbred mice, which are extremely resistantto DEN-induced hepatocarcinogenesis, may carry the Her locifrom the parental DBA/2J mice, but few, if any, of the multiplesensitivity loci. Conversely, the extremely sensitive BXD-11recombinant inbred mice may carry most of the multiple sensitivityloci of the DBA/2J parents, but neither of the major resistanceloci. In order to confirm our genetic model for hepatocarcinogenesisin DBA/2J mice and to evaluate the phenotypic effects of theHcr loci on the Hcs loci of C3H/HeJ mice, we characterized hepatocarcinogensensitivities of F¹ mice generated from the crosses involvingBXD-11, BXD-15, C3H/HeJ and C57BL/ 6J strains. When male micewere initiated with DEN at 12 days of age and liver tumors wereenumerated at 32 weeks of age, (BXD-15xBXD-11)F₁ mice had onesixth the number of liver tumors observed in (C57BL/6JxBXD-11)F₁mice, consistent with our previous conclusion that DBA/2J micepossess hepatocarcinogen resistance genes in spite of theirhigh susceptibility to DEN. Significantly, (C57BL/l6JXC3H/HeJ)F₁mice also had a 2.3-fold greater number of liver tumors and5.5-fold higher total volume of initiated lesions per liveras compared with (BXD-15XC3H/HeJ)F₁ mice, indicating that thehepatocarcinogen resistance genes inherited by BXD-15 mice arecapable of suppressing the Hcs phenotype. Thus the Hcr locimay influence a wide variety of hepatocarcinogen sensitivityloci and be able to act as general resistance loci for chemicalhepatocarcinogenesis. Stereological analysis of initiated hepato-cellularlesions with glucose 6-phosphatase deficiency revealed thatthe resistance genes largely influence the promotion stage ofhepatocarcinogenesis.     

Modulation of natural killer activity by 12-O-tetradecanoylphorbol-13-acetate and benzoyl peroxide in phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice

Following two weeks of topical application of 12-O-tetra-decanoylphorbol-13-acetate(TPA)at 2, 4 and 8 µg/mouse on alternate days (7 x total) orbenzoyl peroxide (BZP) at 10, 20 and 40 mg/mouse, natural killer(NK) activity was determined in local (lymph nodes drainingthe lower dorsal region)n and systemic (spleen) lymphoid tissuein phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice.SENCAR mice, sensitive to tumor induction by TPA in two-stagechemical-induced carcinogenesis protocols, demonstrated suppressionof NK activity in the spleen (no significant change in lymphnodes) and substantial dose-dependent increases in cell numbersin these organs after topical exposure to TPA. B6C3F1 (C57BL/6x C3H Fl) mice, reported to be resistant to TPA-induced promotion,demonstrated significant increases in NK activity in lymph nodes/spleenwith an increase in cell numbers in the draining nodes only.Unlike the C57BL/6 parental Strain, B6C3F1 mice are also reportedto be resistant to promotion with BZP. Significantly, studiesin this laboratory indicated that B6C3F1 mice dosed with BZPdemonstrated increased NK activity in the spleen as was observedafter dosing with TPA. These data suggest that alterations inNK activity as a result of exposure to tumor promoters may,in part, account for the resistance of particular strains ofmice to tumor development. In both SENCAR and B6C3F1 mice, theblastogenic response of spleen cell suspensions isolated fromTPA-dosed animals to phytohemagglutinin (PHA), a T cell lectin,was suppressed in a dose-dependent manner; BZP had no effecton spleen cell responses in either strain. Blastogenic responsesof lymph node cells to PHA were enhanced in both strains ofmice after topical application of TPA and BZP. Therefore, alterationsin lymphoid cell responsiveness to PHA appeared unrelated tothe reported sensitivities of SENCAR and B6C3F1 mice to tumorpromotion.     

Comparative tumorigenicity of 6-nitrochrysene and its metabolites in newborn mice

The tumorigenic activities of 6-nitrochrysene and its metaboliteswere evaluated in the newborn mouse model. Groups of mice weretreated with the appropriate compounds in DMSO by i.p. injectionson the 1st, 8th and 15th day of life. Seven hundred nmol/mouseof 6-nitrochrysene induced significant incidences and multiplicitiesof lung tumors in both sexes; only males were susceptible toliver tumor induction. At 100 nmol/mouse, 6-nitrochrysene hadsignificant tumori-genicity in both lung and liver but was lessactive than at the higher dose. Administration of 100 nmol/mouseof 6-nitrochrysene, only on day 1, caused about the same tumoryield as was observed after treatment with 700 nmol/mouse givenover 3 days. Among the metabolites of 6-nitrochrysene whichwere tested at 100 nmol/mouse, 6-nitrosochrysene and 6-aminochrysenewere significantly less active in the lung and in the liverthan 6-nitrochrysene. In the lung and trans-1, 2-dmydro-ld1hydxy-6-nitn chrysene and trans-l, 2-dihydro-l, 2-dihydroxy-6-aminochysenehad activities comparable to those observed in mice treatedwith equimolar doses of 6-nitrochrysene. In the liver, trans-l,2-dihydro-l, 2-dihydroxy-6-nitrochrysene was more active than6-nitrochrysene based on the number of tumors per mouse. Theseobservations support our hypothesis that 6-nitrochrysene ismetabolically activated by ring-oxidation to trans-1, 2-dihydro-1,2-dihydroxy-6-nitrochrysene, followed by nitro-reduction totrans-1, 2-dihydro-1, 2-dihydroxy-6-amino-chrysene and, finally,oxidation to a diol-apoxide.     

Rapid induction of skin tumors in human but not mouse c-Ha-ras proto-oncogene transgenic mice by chemical carcinogenesis

The rasH2 transgenic mice carry human c-Ha-ras proto-oncogene, and are highly susceptible to chemical carcinogenesis. Previous studies showed that the mutation of c-Ha-ras induced by DMBA in the tumors of rasH2 were detected only in transgenes. To examine if the difference between the codons of the c-Ha-ras gene in human and mouse contributed to the tissue-specific sensitivity to DMBA, we generated a line of transgenic mice, mras, carrying mouse c-Ha-ras genome with its own promoter. Western blot analysis showed that the protein expression of H-RAS in the skin was increased in both rasH2 and mras compared with wild-type. Chemical skin carcinogenesis was induced by DMBA and TPA. In rasH2 mice, the latency of tumor formation was shorter than wild-type littermates. Both the number and the volume of skin tumors were increased in rasH2 than those of wild-type. However, in mras mice, enhancement of tumor formation was not observed as compared with wild-type. The mean number of tumors and the latency of tumor development was almost the same between mras and wild-type littermates. Mutational analysis showed only A to T transversion in human c-Ha-ras transgenes at codon 61 but not in murine endogenous c-Ha-ras gene in the tumors of rasH2. In the tumors of wild-type littermates and mras, A to T transversion in murine c-Ha-ras at codon 61 were detected. These results indicate that the differences in the codon of the c-Ha-ras gene between mouse and human might contribute to the tissue-specific sensitivity of DMBA.     

Ras mutations in methyiclofenapate-induced B6C3F1 and C57BL/1OJ mouse liver tumours

The majority of genotoxic carcinogen-induced liver tumours ofthe sensitive B6C3F1 mouse contain activated H-ras oncogenes.Such mutations also occur in hepatocarcinogenesis resistantstrains. In order to determine whether this is true of non-genotoxiccarcinogen-induced tumours, liver tumours induced in B6C3F1and C57BL/10J mice by methylclofena pate (MCP) were compared.Polymerase chain reaction (PCR) analysis revealed H-ras codon61 mutations in 11/46 B6C3F1 and 4/31 C57BL/10J liver tumours.The nude mouse tumorigenicity (NMT) assay was used to analysetumours without codon 61 mutations. Of the 12 B6C3F1 liver tumourDNAs subjected to this assay, one contained a H-ras codon 117mutation. Further PCR analysis on frozen tumour samples (46B6C3F1 and 15 C57BL/10J) revealed no codon 12 mutations; oneadditional codon 117 mutation was identified in a B6C3F1 tumour.Overall, then, H-ras codon 61 mutations were detected in MCP-inducedB6C3F1 tumours less frequently than in genotoxin-induced tumours.Two B6C3F1 tumours contained codon 117 mutations similar tothose previously found in tumours induced by ciprofibrate, furanand furfural, and in at least one spontaneous tumour. Ras mutationswere also detected in some C57BL/10J tumours, providing furtherevidence that ras oncogenes can participate in hepatocarcinogenesisin resistant mice.